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auto_assembly_v8_d2r.pl
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auto_assembly_v8_d2r.pl
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#!/usr/bin/perl
=head1 Usage
perl auto_assembly.pl [options] <sample_list> <reads_path>
=head1 Example
perl auto_assembly.pl -outdir output -step 0123456789 sample.list /home/xzhong/working_data_01/01_Assembly/37_pipeline
##### each step description #####
step 0: bbnorm.sh;
step 1: cutadapt.sh;
step 2: spades.sh;
step 3: flash.sh or bbmerge.sh[default];
step 4: velveth.sh & velvetg.sh & nucmer.sh & contigs_check.pl;
step 5: chloe.sh for denovo assembly, or carry on reference-guided assembly;
step 6: fill gaps by gapfiller;
step 7: bwa;
step 8: pilon;
step 9: statistics.
#################################
=cut
use strict;
use Getopt::Long;
use File::Basename;
#use FinBin qw($Bin);
my $Bin="/home/xzhong/working_data_01/01_Assembly/37_pipeline";
my ($Step, $Outdir, $Help);
my ($Maxinputreads, $Maxoutputreads);
my ($Adapter1, $Adapter2);
my $Merge;
my ($Mink, $Maxk, $Stepk, $Coverlist,$Refer_DB,$Insert_size,$Insert_size_SD,$Exp_cover,$Contigs_len_cutoff);
my $Clean;
GetOptions(
"outdir:s"=>\$Outdir,
"step:s"=>\$Step,
"maxinputreads:i"=>\$Maxinputreads,
"maxoutputreads:i"=>\$Maxoutputreads,
"adapter1:s"=>\$Adapter1, #adapter1, Sequence of an adapter that was ligated to the 3' end.
"adapter2:s"=>\$Adapter2, #adapter2, 3' adapter to be removed from second read in a pair.
"merge:s"=>\$Merge,
"mink:i"=>\$Mink,
"maxk:i"=>\$Maxk,
"stepk:i"=>\$Stepk, #Velvet will then hash from k=m to k=M with a step of s.
"coverlist:s"=>\$Coverlist, #Velvet's -cov_cutoff.
"insertsize:i"=>\$Insert_size,
"insertsizesd:i"=>\$Insert_size_SD,
"expectedcover:i"=>\$Exp_cover,
"referdb:s"=>\$Refer_DB,
"contigslen:s"=>\$Contigs_len_cutoff, # LSG,SSG and IR 's length cutoff(Kb).
"clean:s"=>\$Clean,
"help!"=>\$Help
);
$Outdir ||=".";
#$Step ||='0123456789';
$Maxinputreads ||=-1;
$Maxoutputreads ||=-1;
$Adapter1 ||="AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC";
$Adapter2 ||="AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT";
$Merge ||="bbmerge";
$Mink ||=51;
$Maxk ||=115;
$Stepk ||=20;
$Refer_DB ||="$Bin/ncbi_cp.fa";
$Insert_size ||=350;
$Insert_size_SD ||=$Insert_size*1/10;
$Exp_cover ||=30;
$Coverlist ||="10,15,7,20";# print "$Coverlist\n";
my @Cover=split(/\,/,$Coverlist);
$Contigs_len_cutoff ||="74,92,10,22,18,29";# "Kb", LSC/SSC/IR
$Clean ||="no";
die `pod2text $0` if (@ARGV == 0 || $Help);
mkdir($Outdir) unless(-d $Outdir);
my $rundir=`pwd`;
chomp $rundir;#print "$rundir";
$Outdir="$rundir/$Outdir"; #print "$Outdir\n";
#`cp $Refer_DB $Outdir/Cp_DB_updated.fa`;
my $sample_list=shift;
my $reads_dir=shift;
`rm "$Outdir/denovo_pass_id.log"` if (-e "$Outdir/denovo_pass_id.log");
`rm "$Outdir/guided_pass_id.log"` if (-e "$Outdir/guided_pass_id.log");
`rm "$Outdir/both_fail_id.log"` if (-e "$Outdir/both_fail_id.log");
open IN, "$sample_list" || die "fail $sample_list";
while(<IN>){
chomp;
my $sample="$_";
#print "$sample\n";
mkdir("$Outdir/$sample") unless(-d "$Outdir/$sample");
## run bbnorm.sh ##
if ($Step=~/0/){
`ls $reads_dir/$sample\_*\_R1*.fastq.gz $reads_dir/$sample\_*\_R2*.fastq.gz > tmp`;
open IN1, "tmp" || die "$!\n";
my $input1=<IN1>; chomp $input1;
my $input2=<IN1>; chomp $input2;
#print "$input1\t$input2\n";
close IN1;
my $bbnorm_shell="$Outdir/$sample/$sample.bbnorm.shell";
open OUT, ">$bbnorm_shell" || die "fail $bbnorm_shell";
print OUT "$Bin/bbnorm.sh in=$input1 in2=$input2 tablereads=$Maxinputreads reads=$Maxoutputreads zerobin=t prefilter=t maxdepth=1000 lowbindepth=10 highbindepth=500 hist=$Outdir/$sample/$sample\_bbnorm.hist out=$Outdir/$sample/$sample\_bbnorm.1.fastq.gz out2=$Outdir/$sample/$sample\_bbnorm.2.fastq.gz outt=$Outdir/$sample/$sample\_bbnorm_excluded.fastq.gz\n";
close OUT;
`rm tmp`;
`sh $bbnorm_shell 1>$bbnorm_shell.log 2>$bbnorm_shell.err`;
}
## run cutadapt.sh ##
if ($Step=~/1/){
my $cutadapter_shell="$Outdir/$sample/$sample.cutadapter.shell";
open OUT1, ">$cutadapter_shell" || die "fail $cutadapter_shell";
print OUT1 "$Bin/cutadapt -a $Adapter1 -A $Adapter2 -o $Outdir/$sample/$sample\_trimmed.1.fastq.gz -p $Outdir/$sample/$sample\_trimmed.2.fastq.gz $Outdir/$sample/$sample\_bbnorm.1.fastq.gz $Outdir/$sample/$sample\_bbnorm.2.fastq.gz\n";
close OUT1;
`sh $cutadapter_shell 1>$cutadapter_shell.log 2>$cutadapter_shell.err`;
}
## run spades.sh ##
if ($Step=~/2/){
my $spades_shell="$Outdir/$sample/$sample.spades.shell";
open OUT1x, ">$spades_shell" || die "fail $spades_shell";
print OUT1x "$Bin/python $Bin/spades.py --only-error-correction --careful --pe1-1 $Outdir/$sample/$sample\_trimmed.1.fastq.gz --pe1-2 $Outdir/$sample/$sample\_trimmed.2.fastq.gz -o $Outdir/$sample/spades_outdir\n";
close OUT1x;
`sh $spades_shell 1>$spades_shell.log 2>$spades_shell.err`;
}
## run flash.sh ##
if($Step=~/3/ && ($Merge ne "bbmerge")){
my $flash_shell="$Outdir/$sample/$sample.flash.shell";
open OUT2, ">$flash_shell" || die "fail $flash_shell";
print OUT2 "$Bin/flash -q -m 10 -x 0.2 --max-overlap 125 -z -d $Outdir/$sample -o $sample $Outdir/$sample/spades_outdir/corrected/$sample\_trimmed.1.fastq.00.0_0.cor.fastq.gz $Outdir/$sample/spades_outdir/corrected/$sample\_trimmed.2.fastq.00.0_0.cor.fastq.gz\n";
close OUT2;
`sh $flash_shell 1>$flash_shell.log 2>$flash_shell.err`;
}
## run bbmerge.sh [default] ##
if($Step=~/3/ && ($Merge eq "bbmerge")){
my $bbmerge_shell="$Outdir/$sample/$sample.bbmerge.shell";
open OUT2x, ">$bbmerge_shell" || die "fail $bbmerge_shell";
print OUT2x "$Bin/bbmerge.sh in1=$Outdir/$sample/spades_outdir/corrected/$sample\_trimmed.1.fastq.00.0_0.cor.fastq.gz in2=$Outdir/$sample/spades_outdir/corrected/$sample\_trimmed.2.fastq.00.0_0.cor.fastq.gz out=$Outdir/$sample/$sample.extendedFrags.fastq.gz outu1=$Outdir/$sample/$sample.notCombined_1.fastq.gz outu2=$Outdir/$sample/$sample.notCombined_2.fastq.gz";
close OUT2x;
`sh $bbmerge_shell 1>$bbmerge_shell.log 2>$bbmerge_shell.err`;
}
## run velveth.sh & velvetg.sh & nucmer.sh & contigs_check.pl ##
if($Step=~/4/){
open OUTa, ">$Outdir/$sample/cover_kvalue.list" || die "$!\n";
lable: for(my $j=0; $j<@Cover; $j++){
#print "$Cover[$j]\n";
my $velveth_shell="$Outdir/$sample/$sample\_$Cover[$j]\_velveth_shell";
open OUT3, ">$velveth_shell" || die "fail $velveth_shell";
print OUT3 "$Bin/velveth $Outdir/$sample/$sample\_$Cover[$j] $Mink,$Maxk,$Stepk -shortPaired -fastq.gz -separate $Outdir/$sample/$sample.notCombined_1.fastq.gz $Outdir/$sample/$sample.notCombined_2.fastq.gz -long $Outdir/$sample/$sample.extendedFrags.fastq.gz -short $Outdir/$sample/spades_outdir/corrected/$sample\_trimmed.00.0_0.cor.fastq.gz\n";
close OUT3;
`sh $velveth_shell 1>$velveth_shell.log 2>$velveth_shell.err`;
my $addnum=int(($Maxk-$Mink)/$Stepk);# caculate No. of times to increase k values.
my $kvalue=$Mink;
for(my $i=0; $i<=$addnum; $i++){
print OUTa "$sample\_$Cover[$j]\_$kvalue\n";
my $velvetg_shell="$Outdir/$sample/$sample\_$Cover[$j]\_$kvalue\_velvetg_shell";
open OUT4, ">$velvetg_shell" || die "fail $velvetg_shell";
print OUT4 "$Bin/velvetg $Outdir/$sample/$sample\_$Cover[$j]\_$kvalue -ins_length $Insert_size -ins_length_sd $Insert_size_SD -cov_cutoff $Cover[$j] -exp_cov $Exp_cover -min_contig_lgth 2000 -scaffolding yes -clean yes\n";
close OUT4;
`sh $velvetg_shell 1>$velvetg_shell.log 2>$velvetg_shell.err`;
my $contigs_dir="$Outdir/$sample/$sample\_$Cover[$j]\_$kvalue";
unless (-z "$contigs_dir/contigs.fa"){
`perl $Bin/fasta_shorter.pl $contigs_dir/contigs.fa "$sample\_$Cover[$j]\_$kvalue" > $contigs_dir/contigs.lable.fa`;
`perl $Bin/align_draw.pl -refer $Outdir/Cp_DB_updated.fa -query $contigs_dir/contigs.lable.fa -fig no -coverlen 800 -coverrate 6 -outdir $contigs_dir`;
unless (-z "$contigs_dir/Cp_DB_updated.fa_vs_contigs.lable.fa.coords.cp.id"){
`perl $Bin/choose_refer_contigs_v2.pl $contigs_dir/Cp_DB_updated.fa_vs_contigs.lable.fa.coords.cp.id |$Bin/msort -k nr15 >$contigs_dir/refer_contigs_denovo.stat`;
open INc, "$contigs_dir/refer_contigs_denovo.stat" || die "$!\n";
my $bestr=<INc>;
chomp($bestr);
close INc;
my @info=split(/\s+/, $bestr);
my $bestrefer=$info[0];
`less $contigs_dir/Cp_DB_updated.fa_vs_contigs.lable.fa.coords.cp.id |grep "$bestrefer" >$contigs_dir/refer_contigs_denovo_best.align`;
`perl $Bin/contigs_check.pl $contigs_dir/refer_contigs_denovo_best.align $Contigs_len_cutoff > tmp`;
open IN2, "tmp" || die "$!\n";
$_=<IN2>;
`rm tmp`;
if(/PASS/){
my $pass_path="$Outdir\/$sample\/denovo_pass_path.log";
open OUT8, ">$pass_path" || die "$!\n";
print OUT8 "$Outdir/$sample\t$sample\_$Cover[$j]\_$kvalue\t$bestrefer\n";
close OUT8;
last lable;
}
close IN2;
}
}
$kvalue +=$Stepk;
}
}
close OUTa;
}
## run chloe.sh ##
if($Step=~/5/){
#open INa, "$Outdir/$sample/cover_kvalue.list" || die "$!\n";
#while(<INa>){
# chomp;
# if (-e "$Outdir/$sample/$_/Cp_DB_updated.fa_vs_contigs.lable.fa.coords.cp.id"){
# unless (-z "$Outdir/$sample/$_/Cp_DB_updated.fa_vs_contigs.lable.fa.coords.cp.id"){
# `cat $Outdir/$sample/$_/Cp_DB_updated.fa_vs_contigs.lable.fa.coords.cp.id >>$Outdir/$sample/Cp_DB_updated.fa_vs_all.contigs.lable.fa.coords.cp.id`;
# }
# }
#}
#close INa;
#if (-e "$Outdir/$sample/Cp_DB_updated.fa_vs_all.contigs.lable.fa.coords.cp.id"){
# `perl $Bin/choose_refer_contigs.pl $Outdir/$sample/Cp_DB_updated.fa_vs_all.contigs.lable.fa.coords.cp.id |$Bin/msort -k nr13 >$Outdir/$sample/refer_contigs_guided.stat`;
#print "$Outdir/$sample/denovo_pass_path.log\n";
open INb, "$Outdir/$sample/denovo_pass_path.log" || die "$!\n";
my $bestrc=<INb>;
chomp($bestrc);
#print "$bestrc\n";
close INb;
my @info=split(/\s+/, $bestrc);
my @info2=split(/\//, $info[0]);
my $bestcontigs=$info2[-1];
print "$bestcontigs\n";
#`less $Outdir/$sample/Cp_DB_updated.fa\_vs_all.contigs.lable.fa.coords.cp.id |grep "$bestrefer" |grep "$bestcontigs" >$Outdir/$sample/refer_contigs_guided_best.align`;
`perl $Bin/link_contigs_v3.pl $Outdir/$sample/$bestcontigs/contigs.lable.fa $Outdir/$sample/$bestcontigs/refer_contigs_denovo_best.align Cp_DB $bestcontigs $Outdir/$sample`;
`perl $Bin/delete_overlap.pl $Outdir/$sample/$bestcontigs\_linked_cp.fa >$Outdir/$sample/$bestcontigs\_linked_cp_delete.fa`;
`perl $Bin/bwa_perl.pl -ref $Outdir/$sample/$bestcontigs\_linked_cp_delete.fa -pelist $Outdir/$sample/spades_outdir/corrected/$sample\_bbnorm.1.fastq.00.0_0.cor.fastq.gz,$Outdir/$sample/spades_outdir/corrected/$sample\_bbnorm.2.fastq.00.0_0.cor.fastq.gz -sample $bestcontigs\_1st -outdir $Outdir/$sample`;
`sh $Outdir/$sample/$bestcontigs\_1st_bwa.sh 1>$Outdir/$sample/$bestcontigs\_1st_bwa.sh.log 2>$Outdir/$sample/$bestcontigs\_1st_bwa.sh.err`;
`java -jar $Bin/pilon-1.16.jar --genome $Outdir/$sample/$bestcontigs\_linked_cp_delete.fa --frags $Outdir/$sample/$bestcontigs\_1st.pe0.sort.bam --output $Outdir/$sample/$sample\_guided_1st_pilon 1>$Outdir/$sample/$sample\_guided_1st_pilon.log 2>$Outdir/$sample/$sample\_guided_1st_pilon.err`;
}
## run gapfiller ##
if ($Step=~/6/){
my $final_fa; my $after_fill_gap;
#$final_fa="$Outdir/$sample/$sample\_denovo_Chloe.fa" if(-e "$Outdir/$sample/$sample\_denovo_Chloe.fa");
$final_fa="$Outdir/$sample/$sample\_guided_1st_pilon.fasta" if (-e "$Outdir\/$sample\/$sample\_guided_1st_pilon.fasta");
if (-e $final_fa){
`perl $Bin/fa_quality.pl -Head -len -gap -N -gc $final_fa >$final_fa.stat`;
open INd,"$final_fa.stat" || die "$!\n";
<INd>;
$_=<INd>;
my @info=split(/\s+/, $_);
close INd;
if ($info[2] !=0){
open OUT12,">$Outdir/$sample/lib.guided.txt" || die "$!\n";
print OUT12 "$sample\_PE bwa $Outdir/$sample/spades_outdir/corrected/$sample\_bbnorm.1.fastq.00.0_0.cor.fastq.gz $Outdir/$sample/spades_outdir/corrected/$sample\_bbnorm.2.fastq.00.0_0.cor.fastq.gz $Insert_size 0.25 FR\n";
close OUT12;
open OUT13,">$Outdir/$sample/gapfiller.guided.sh" || die "$!\n";
print OUT13 "cd $Outdir/$sample;\nperl /home/xzhong/Software/GapFiller/GapFiller.pl -l $Outdir/$sample/lib.guided.txt -s $final_fa -b gap_filling_guided;\ncd $rundir;\n";
close OUT13;
`sh $Outdir/$sample/gapfiller.guided.sh 1>$Outdir/$sample/gapfiller.guided.sh.log 2>$Outdir/$sample/gapfiller.guided.sh.err`;
$after_fill_gap="$Outdir/$sample/gap_filling_guided/gap_filling_guided.gapfilled.final.fa";
}else{
$after_fill_gap=$final_fa;
}
`perl $Bin/end_check.pl $after_fill_gap >$Outdir/$sample/after_fill_gap_check_guided.fa`;
}
}
## run bwa ##
if($Step=~/7/){
if (-e "$Outdir\/$sample\/$sample\_guided_1st_pilon.fasta"){
my $final_fa="$Outdir/$sample/after_fill_gap_check_guided.fa";
`perl $Bin/bwa_perl.pl -ref $final_fa -pelist $Outdir/$sample/spades_outdir/corrected/$sample\_bbnorm.1.fastq.00.0_0.cor.fastq.gz,$Outdir/$sample/spades_outdir/corrected/$sample\_bbnorm.2.fastq.00.0_0.cor.fastq.gz -sample $sample\_2nd -outdir $Outdir/$sample`;
`sh $Outdir/$sample/$sample\_2nd_bwa.sh 1>$Outdir/$sample/$sample\_2nd_bwa.sh.log 2>$Outdir/$sample/$sample\_2nd_bwa.sh.err`;
}
}
## run pilon ##
if($Step=~/8/){
if (-e "$Outdir\/$sample\/$sample\_guided_1st_pilon.fasta"){
my $final_fa="$Outdir/$sample/after_fill_gap_check_guided.fa";
open OUT11, ">$Outdir/$sample/$sample\_pilon_guided.sh" || die "$!\n";
print OUT11 " $Bin/java -jar $Bin/pilon-1.16.jar --genome $final_fa --frags $Outdir/$sample/$sample\_2nd.pe0.sort.bam --output $Outdir/$sample/$sample\_pilon_guided\n";
close OUT11;
`sh $Outdir/$sample/$sample\_pilon_guided.sh 1>$Outdir/$sample/$sample\_pilon_guided.sh.log 2>$Outdir/$sample/$sample\_pilon_guided.sh.err`;
`perl $Bin/end_restore.pl $Outdir/$sample/$sample\_pilon_guided.fasta >$Outdir/$sample/$sample\_pilon\_guided_restore.fasta`;
open OUTcp, ">$Outdir/$sample/$sample\_guided_permutation.sh" || die "$!\n";
print OUTcp "$Bin/java -jar $Bin/CircularPermutation.jar $Outdir/$sample/$sample\_pilon\_guided_restore.fasta $Bin/NC_030504_refs.fa $Outdir/$sample/$sample\_pilon\_guided_permutation.fasta\n";
close OUTcp;
`sh $Outdir/$sample/$sample\_guided_permutation.sh 1>$Outdir/$sample/$sample\_guided_permutation.sh.log 2>$Outdir/$sample/$sample\_guided_permutation.sh.err`;
if (-e "$Outdir/$sample/$sample\_pilon\_guided_permutation.fasta"){
`ln -s $Outdir/$sample/$sample\_pilon\_guided_permutation.fasta $Outdir/$sample/$sample\_pilon\_guided_final.fasta`;
}else{
`ln -s $Outdir/$sample/$sample\_pilon\_guided_restore.fasta $Outdir/$sample/$sample\_pilon\_guided_final.fasta`;
}
`perl $Bin/fa_quality.pl -Head -len -gap -N -gc $Outdir/$sample/$sample\_pilon\_guided_final.fasta > $Outdir/$sample/$sample\_pilon\_guided_final\_fasta.stat\n`;
}
# if (-e "$Outdir/$sample/$sample\_denovo_Chloe.fa"){
# `cat $Outdir/Cp_DB_updated.fa $Outdir/$sample/$sample.pilon.final.fasta >$Outdir/Cp_DB_updated.fa.tmp`;
# `mv $Outdir/Cp_DB_updated.fa.tmp $Outdir/Cp_DB_updated.fa`;
# }
}
## run statistics ##
if ($Step=~/9/){
#`rm $Outdir/reads.stat` if (-e "$Outdir/reads.stat");
`perl $Bin/reads_stat.pl $Outdir/$sample/$sample.bbnorm.shell.err $Outdir/$sample/$sample.cutadapter.shell.log $sample >>$Outdir/reads.stat`;
if (-e "$Outdir/$sample/$sample\_denovo_Chloe.fa"){
`perl $Bin/assembly_stat.pl $Outdir/$sample/$sample.pilon.final.fasta.stat $Outdir/$sample/denovo_pass_path.log $sample\_pilon.sh.log $sample denovo >> $Outdir/denovo_assembly.stat`;
}
if (-e "$Outdir\/$sample\/$sample\_guided_1st_pilon.fasta"){
`perl $Bin/assembly_stat2.pl $Outdir/$sample/$sample.pilon.final.fasta.stat $Outdir/$sample/refer_contigs_guided.stat $sample\_pilon.sh.log $sample reference-guided >> $Outdir/refer_assembly.stat`;
}
}
## remove the temporary ##
if ($Clean eq 'yes'){
`rm $Outdir/$sample/$sample\_bbnorm*.gz`;
`rm $Outdir/$sample/$sample\_trimmed*.gz`;
`rm $Outdir/$sample/spades_outdir/corrected/$sample\_trimmed.*.fastq.*.fastq.gz`;
`rm $Outdir/$sample/$sample\_*/Sequences`;
`rm $Outdir/$sample/$sample\_*/Roadmaps`;
`rm $Outdir/$sample/$sample\_*/Graph2`;
`rm $Outdir/$sample/$sample\_*/PreGraph`;
`rm -rf $Outdir/$sample/gap_filling/reads`;
`rm -rf $Outdir/$sample/gap_filling/intermediate_results`;
`rm $Outdir/$sample/*_1st.*.sort.bam`;
}
}
close IN;